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The Arrest of Biological Time as a Bridge to Engineered Negligible Senescence Jerry B. Lemler, M.D., Medical Director Alcor Life Extension Foundation Scottsdale, AZ, USA www.alcor.org

First of all I would like to thank Aubrey for inviting me to give this talk. It is my pleasure to discuss with this audience the concept of putting biological time on hold so as to create a bridge to both engineered negligible senescence and other valuable medical technologies of the future.

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Two Ways to Modify Biological Time Nuclear Transfer — The egg cell serves as a “time machine” to take adult cells back in biological time to potentially form autologous totipotent stem cells. Cryobiology — Very low temperatures serve as a “time machine” to take adult cells forward in time in a changeless state until it is time to use them.

The opponent of the gerontologist is biological time. As you all heard from Michael West earlier in this meeting, one way to conquer biological time has been proposed in the form of nuclear transfer, in which a senescent cell or its nucleus can be effectively taken back in biological time by being transferred to the “time machine” of an egg cell. Equally familiar to this audience is the phenomenon of the time machine provided by cryobiology, in which very low temperatures allow living cells to be moved forward in objective time while standing still in biological time until they are needed.

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An Audacious Proposal: The Concept of “Medical Time Travel” Many diseases and conditions that are incurable today (including senescence!) will probably be curable at some time in the future. If the patients of today were able to benefit from the medical technologies of the future, many terminal patients could probably be rescued. Could cryobiological time arrest allow today’s patients to wait for future medicine?

You all heard yesterday from Aubrey de Grey about his so-called crazy proposal for the elimination of cancer in humans. In the same vein, I now offer you another audacious proposal. The concept of what I’d like to call medical time travel is simple. Most of you would agree that many diseases and symptoms that are not presently curable probably will be curable sometime in the future, and that if patients of today could somehow benefit from the medical technology of the future, many people who are now considered terminal could be rescued. The question we will now consider is, could the time machine of cryobiology conceivably provide a way for today’s patients to wait for future medicine?

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A Basis for Medical “Time Travel” One Fact: For all practical purposes, at approx. -196°C biological changes do not occur over periods of centuries to millennia. One Assumption: Medical technology will continue to improve. After several hundred years, medicine will be able to reverse any injury whose reversal is not forbidden by fundamental physical law. Deduction: If patients can be cooled to approx. -196°C today without producing injury that is irreversible in principle, it should be possible to recover today’s patients with the help of tomorrow’s technology.

We can start to think about this idea by putting together one fact and one assumption. The fact is that for all practical purposes, at -196 degrees centigrade, (the temperature of liquid nitrogen), biological changes do not occur, even over periods lasting centuries to millennia. The assumption is that medical technology will continue to improve for as long as the patient remains at that temperature. It is possible to project that after several hundred years medicine should be able to reverse any injury whose reversal is not forbidden by the laws of physics and chemistry. Therefore, if patients can be stored near liquid nitrogen temperature now without producing injury that is irreversible in principle, then it should be possible eventually to recover today’s patients with the benefit of tomorrow’s technology.

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“Brief Proposal On Immortality: An Interim Solution” by George M. Martin Perspectives in Biology and Medicine 14(2): 339-340, 1971 “…a…solution to the ‘terrible problem of death awareness’…which appeals to me is one which preserves the central nervous system. The spectacular success of cryobiological procedures…suggests…satisfactory…preservation may yet be achieved…in situ….”

You may find it interesting that Dr. George Martin thought the same way in 1971 when he said a solution to the whole problem of death awareness, which appealed to him, is one that preserves the central nervous system, and that the spectacular successes of cryobiological procedures suggest that satisfactory preservation may be achievable in situ.

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Is Today’s Injury Potentially Reversible? Injury category 1: today’s fatal diseases e.g., heart disease, cancer, infectious diseases, the effects of aging Injury category 2: brief anoxia after “clinical death” (necessary under current law) Injury category 3: cryopreservation injury

Of course, this will only work if the injury induced today is potentially reversible. We can identify three fundamental categories of injury to consider, and these are: today’s fatal diseases, the anoxia associated with brief clinical death, and cryopreservation injury. Clearly, the reversal of today’s fatal diseases will require extensive research and time, but should be plausible to this audience, as most of you are willing to seriously consider the prevention and even the reversal of senescence. Consequently we will move on to the more pressing issues of clinical death and cryopreservation injury.

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Understanding “Clinical Death” A legal, not a biological, definition of death Cardiopulmonary arrest Not “brain death”

Under current law it is necessary to have a physician declare legal death before any attempt at cryopreservation can be made. Legal or clinical death can be declared after there has been a short period of cardiopulmonary arrest, after which resuscitation methods can be and routinely are applied. It is important to distinguish this situation from that of brain death, which is in no way required by current law as it pertains to our proposal.

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Surviving Clinical Death Following cardiac arrest, injury will accumulate until a rescue team is able to restore circulation and oxygenation. What is the safe window of time within which a team can act before brain injury becomes irreversible by present technology?

The risk for our proposal is that the brain will be anoxic during the time required for the declaration of death itself, plus any time required for any rescue team to restore circulation and oxygenation to the brain or to perform other stabilizing procedures such as cooling. We therefore need to have an idea of how much time the brain can be subjected to ischemia and still be recovered without permanent injury. In other words, is there a window of opportunity in which rescue teams can act?

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Reversibility of Clinical Death: 1 Survival of Adult Dogs with Normal Mentation Following 14-16 Min of Normothermic Whole-Body Circulatory Arrest Dog Cerberus Scroffy Claudia Maude Bob Stuart Temperature* 35.9 37.3 38.0 37.7 37.9 37.6 Arrest Time** 14.25 min 14.75 min 14.80 min 15.75 min 15.42 min 16.25 min *Tympanic temperature just before fibrillation; degrees Celsius **Time with mean arterial pressure below 30 mmHg

Contrary to the clinical impression that the limit for reversible brain anoxia is only 5 minutes, we know the actual limit in dogs is at least 16 minutes, because the dogs in this table were subjected to up to 16 minutes of normothermic cardiac arrest with no pretreatment and they recovered without any brain damage whatsoever. So we know the old 5 minute limit for reversibility of brain death is in fact not a true biological limit.

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Reversibility of Clinical Death: 2 “Hossmann’s Cat” 60 min of total cerebral ischemia at 37°C was compatible with: Normal ambulation Normal self-cleaning Recognition of lab staff Purring Indefinite survival. K.-A. Hossmann et al., 1987: J Neurol. Sci. 77:305-320.

We also know that 16 minutes is not the limit because of Hossman’s cat. In 1987 his group reported that they had allowed a cat to survive a procedure that completely stopped cerebral blood flow at normal body temperature for one hour. After the procedure the cat recovered and survived indefinitely, was able to walk, clean itself, recognize lab staff, and purr. This experiment was the culmination of years of research by Hossmann and colleagues indicating that brain injury is reversible in cats and monkeys after at least one hour of complete normothermic cerebral ischemia.

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Reversibility of Clinical Death: 3 Rapid Interruption of Warm Ischemic Injury

So the true biological limit is at least 60 minutes, more than enough time for a waiting rescue team to respond. In principle, a patient can be put into an ice bath and cooled externally immediately after legal death is pronounced. Cooling can be assisted by external cardiac compression using one of these Lucas chest compression devices, along with oxygenation given by endotrachial tube, and so the first stages of reversing brain anoxia can be instituted quite rapidly, well within the period of reversibility of brain damage. Even more advanced techniques of rapid cooling are feasible and practiced, further reducing anoxic injury.

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This is a portable perfusion and cooling unit used to induce rapid whole body hypothermia and initiate perfusion and reoxygenation. Note the perfusate reservoirs and all necessary equipment to perform patient stabilization under field conditions.

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And, as you can see in this diagram, the cooling rate you can achieve with these devices is substantial, which further guarantees protection against anoxic injury. The blue line represents the cooling rate achievable by perfusion.

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Surviving Cryopreservation Injury Freezing (ice formation, left) tears tissues Vitrification (glass formation, right) does not Source: Cryobiology 21: 407-426, 1984.

This leaves one remaining potential source of injury, and that’s cryopreservation injury. In the old days cryopreservation used to be done by freezing. Freezing is the formation of ice crystals. Ice crystals appear white when they form in tissues, as you can see in the frozen kidney on the left. Unfortunately, ice crystals also cause major damage to organized tissues, and that’s why we haven’t been using this process since the year 2000 at Alcor. Instead we have been using a process called vitrification, which is the formation of a glass. Glass formation does not permit ice crystals to develop, and therefore there is no ice damage associated with glass formation. On the right you can see that it is possible to vitrify a whole organ, which in this case is a rabbit kidney. This kidney looks the same as a fresh kidney even though it was photographed at 140 degrees below zero.

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This slide explains the concept in more detail. As you gradually increase the concentration of a cryoprotective agent, thereby replacing water, ice formation diminishes. Here we see ice crystals inside a red blood cell getting smaller and smaller and eventually disappearing altogether. At the highest concentration even extracellular ice disappears despite cooling to cryogenic temperatures. This is what we mean by vitrification.

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Recent experiments have shown that this process can actually be applied to an entire brain. This is a scanning electron microscope image of some cerebral cortex removed from a rabbit brain that was vitrified, rewarmed, and fixed by vascular perfusion. The cryoprotectant was then gradually removed. The surface of the brain is at the top, with white matter seen as the rough area at the bottom. You can see a blood vessel near the top that is intact. Other than a few mechanical artifacts, the substance of the cerebral cortex and the underlying white matter is remarkably nice and smooth. There were no ice cavities.

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You can verify this by zooming in. You can see that the apparent holes in the tissue are capillaries. Ice crystals would be massive on this scale, and we don’t see any of them. Therefore, this brain actually did vitrify.

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This is the same cerebral cortex but seen by transmission electron microscopy. The cortical pyramidal cells are osmotically shrunken and distorted but otherwise appear to be intact. You can see shrunken axoplasm inside myelinated nerve fibers. You also see normal-appearing capillaries. There are no ice crystals anywhere to be seen, and as far as we can tell, the ultrastructure is fundamentally preserved. The diagonal gap is an artifact.

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We also looked elsewhere in the brain. This happens to be the hippocampus.

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If you look at the hippocampus either by scanning or transmission electron microscopy, again you see the same thing. No ice anywhere. The tissue is certainly dehydrated, but you do not see structural disintegration, or clearly irreversible damage to the ultrastructure.

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This is an area in the middle of the hippocampus, again showing all of the neuropil compressed and dehydrated, but intact.

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This is the dentate gyrus, where the same picture is seen. This goes on for field after field, slide after slide. I don’t want to bore you with it, but no matter where we look we couldn’t see any evidence of ice formation, but we do see preserved capillaries, shrunken cells, and intact neuropil.

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Is Cryoprotectant Toxicity Fundamentally Irreversible? Whole rabbit kidneys support life after transplantation following perfusion with vitrification solutions. Rat hippocampal slices survive vitrification and rewarming.

So it looks like we can avoid the mechanical effects of ice in vitrifying the brain, but what about the toxicity of the agents that are needed to replace water? Do cryoprotectants have a toxic effect that’s irreversible in principle? We think that’s unlikely, for a couple of reasons. For one thing, whole rabbit kidneys were recently reported to support life after transplantation despite perfusion with an ultrastable vitrification solution, M22, and were able to bring serum creatinine levels down to normal. So if the damage caused by these cryoprotectants is reversible spontaneously today it doesn’t seem likely that it will be irreversible with the benefit of better technology in the future. The other reason has to do with the brain itself. In particular, studies currently being prepared for publication have shown that rat hippocampal slices can actually survive vitrification and re-warming.

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Conclusion The major hazards of attempted medical time travel Contemporary diseases Cerebral ischemia Mechanical tissue disruption by ice, & Toxic damage from vitrification solutions all seem unlikely to require remedies that are impossible according to physical law.

On the basis of all of this evidence, it seems to us that the major hazards of attempted medical time travel, including diseases, ischemia, tissue disruption by ice, and stress from vitrifying solutions, all seem unlikely to require remedies that are incompatible with physical law.

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… and therefore it is justified to regard attempted medical time travel as a conservative approach to the treatment of otherwise terminally ill patients.

And therefore we believe it is justified to regard attempted medical time travel as a conservative approach to otherwise terminally ill patients.

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Reaching the Limits of Physical Law: Medical Nanotechnology

If anyone would like to know more about how reaching the limits of physical law in medicine might be achieved, I would be happy to refer you to several scholarly works on the subject, such as these particularly outstanding books.

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www.alcor.org

In the meantime, thank you very much for your attention.